The invention relates to methods and compositions for modulating ubiquitin dependent proteolysis.
Ubiquitin-dependent proteolysis is a key regulatory mechanism that controls diverse cellular processes (reviewed in Hochstrasser 1996). In this pathway, ubiquitin is transferred via transthioesterification along a cascade of carrier enzymes, E1xe2x86x92E2xe2x86x92E3, and ultimately conjugated in an isopeptide linkage to a lysine residue of a substrate protein. Reiteration of the ubiquitin transferase reaction results in formation of a polyubiquitin chain on the substrate, which is then recognized by the 26S proteasome, and rapidly degraded. Specificity in protein ubiquitination derives from E3 enzymes, also known as ubiquitin-ligases (Hershko et al. 1983). In some cases, an E3 facilitates recognition of the target protein by an E2, while in others an E3 accepts a ubiquitin thioester from an E2 and directly transfers ubiquitin to the substrate (Scheffner et al. 1995). Although substrate recognition is a key aspect of ubiquitin dependent proteolysis, the identification of E3 enzymes has been problematic because the few known E3 families bear no sequence relationship to each other.
Ubiquitin-dependent proteolysis is essential for two major cell cycle transitions, the G1 to S phase transition and the metaphase to anaphase transition (reviewed in King et al. 1996). These transitions mediate alteration between states of high and low cyclin-dependent kinase (Cdk) activity, which in turn ensures that DNA replication origins fire only once per cell cycle and that chromosome segregation follows DNA replication (reviewed in Nasmyth 1996). Key targets of the ubiquitin proteolytic pathway at these transitions include positive regulators of Cdks, the cyclins, and negative regulators of Cdks, the Cdk inhibitors. In budding yeast, a single Cdk, Cdc28 (or Cdk1) is activated in G1 phase by the G1 cyclins Cln1-Cln3, and in S through M phase by the mitotic cyclins, Clb1-Clb6 (reviewed in Nasmyth, 1996). A motif called the destruction box targets mitotic cyclins and other proteins to a cell cycle-regulated E3 ubiquitin-ligase called the Anaphase Promoting Complex (APC) or cyclosome (reviewed in King et al. 1996). In contrast, phosphorylation targets G1 cyclins and Cdk inhibitors for degradation via a constitutive ubiquitination pathway (reviewed in Deshaies, 1997). Genetic analysis in budding yeast has revealed several components of this pathway: Cdc4, a WD40 repeat protein (Yochem and Byers, 1987), Cdc34, an E2 ubiquitin conjugating enzyme (Goebl et al. 1988), Cdc53 a protein that forms a tight complex with phosphorylated Clns (Willems et al. 1996), Grr1, a leucine rich repeat protein (Flick and Johnston, 1981), and Skp1, a protein that binds to a motif called the F-box (Bai et al. 1996). The F-box motif occurs in Cdc4, Grr1 and several other yeast and mammalian proteins (Bai et al. 1996). Cells lacking functional Cdc4, Cdc34, Cdc53 or Skp1 arrest in G1 because the Cdk inhibitor Sic1 is not degraded, which prevents the onset of Clb-Cdc28 activity and initiation of DNA replication (Nugroho and Mendenhall 1994; Schwob et al. 1994; Bai et al. 1996). In late G1 phase, Sic1 is phosphorylated by the Cln-Cdc28 kinases and thus targeted for ubiquitin dependent proteolysis (Schwob et al. 1994; Schneider et al. 1996; Tyers 1996). Recently, a requirement for Cdc4, Cdc34 and Cln2-Cdc28 activity in Sic1 ubiquitination has been demonstrated in an in vitro yeast extract system (Verma et al. 1997). Cdc34, Cdc53 and Skp1 are also required for Cln degradation (reviewed in Deshaies 1997), as is Grr1 (Barral at al. 1995), although this protein was originally identified because of its role in glucose repression (Flick and Johnston 1991).
Other important regulatory proteins are degraded via the Cdc34 pathway, including the Cln-Cdc28 inhibitor Far1 (McKinney et al. 1993; Henchoz et al. 1997), the replication protein Cdc6 (Piatti et al. 1996), and the transcription factor Gcn4 (Kornitzer et al. 1994). Aside from its G1 function, Skp1 also plays a role in G2 because certain conditional alleles of SKP1 arrest cells in G2, and because Skp1 is a component of the Cbf3 kinetochore complex (Bai et al. 1996; Connelly and Hieter 1996; Stemmann and Lechner 1996).
Genetic and biochemical evidence indicates that Cdc53 interacts with Cdc4 and Cdc34 (Willems et al. 1996; Mathias et al. 1996), and that the F-box of Cdc4 binds Skp1 (Bai et al. 1996). These interactions, and the fact that Cdc53 physically associates with phosphorylated forms of Cln2, suggest that Cdc4, Cdc34, Cdc53 and Skp1 may participate in an E2/E3 ubiquitination complex that recognizes and ubiquitinates phosphorylated substrates (Bai et al. 1996; Willems et al. 1996). Divergence of the Sic1 and Cln degradation pathways apparently occurs at the level of the two F-box proteins. Cdc4 is required for degradation of Sic1 (Schwob et al. 1994), whereas Grr1 is required for Cln1/2 degradation (Barral et al. 1995). It was therefore hypothesized that distinct F-box proteins recruit specific substrates to an E3 ubiquitin-ligase complex that contains Skp1 (Bai et al. 1996). The existence of a complex in vivo containing F-box proteins and Cdc34, Cdc53 and Skp1 has yet to be demonstrated.
Through analysis of Cdc53-interacting proteins the present inventors determined that Cdc53 forms complexes with Skp1, Cdc34, and each of the F-box proteins Cdc4, Grr1 and Met30 in vivo. Each F-box protein confers functional specificity on a core Cdc34-Cdc53-Skp1 complex for Sic1 degradation, Cln degradation and methionine biosynthesis gene regulation, respectively. The present inventors showed that Cdc53 is a scaffold that tethers Skp1/F-box proteins to Cdc34 within an E2/E3 ubiquintination complex. The present inventors have also identified a specific region on Cdc53 that binds to Skp1.
Broadly stated the present invention relates to (a) a complex comprising an E2 ubiquitin conjugating enzyme, a protein of the Cullin family, an F-box binding protein, and optionally a protein containing an F-box motif; and (b) a complex comprising a protein of the Cullin family and a protein containing an F-box motif. The invention is also directed to (a) a peptide derived from the binding domain of an E2 ubiquitin conjugating enzyme that interacts with a protein of the Cullin family; (b) a peptide derived from the binding domain of a protein of the Cullin family that interacts with an E2 ubiquitin conjugating enzyme; (c) a peptide derived from the binding domain of a protein of the Cullin family that interacts with an F-box binding protein; preferably a peptide of the formula I or Ia or (d) a peptide derived from the binding domain of an F-box binding protein that interacts with a protein of the Cullin family. The invention also contemplates antibodies specific for the complexes and peptides of the invention.
The present invention also provides a method of modulating ubiquitin dependent proteolysis comprising administering an effective amount of one or more of the following: (a) a complex comprising an E2 ubiquitin conjugating enzyme, a protein of the Cullin family, an F-box binding protein, and optionally a protein containing an F-box motif; (b) a complex comprising a protein of the Cullin family and a protein containing an F-box motif; (c) a peptide derived from the binding domain of an E2 ubiquitin conjugating enzyme that interacts with a protein of the Cullin family; (d) a peptide derived from the binding domain of a protein of the Cullin family that interacts with an E2 ubiquitin conjugating enzyme; (d) a peptide derived from the binding domain of a protein of the Cullin family that interacts with an F-box binding protein; preferably a peptide of the formula I or Ia (e) a peptide derived from the binding domain of an F-box binding protein that interacts with a protein of the Cullin family; or (f) enhancers or inhibitors of the interaction of an E2 ubiquitin conjugating enzyme or an F-box binding protein, with a protein of the Cullin family.
In a preferred embodiment of the invention a method is provided for modulating ubiquitin dependent proteolysis comprising administering an effective amount of one or more of the following: (a) a complex comprising Cdc34-Cdc53-Skp1; (b) a complex comprising Cdc34-Cdc53-Ckp1-protein containing an F-box motif; (c) a complex comprising Cdc53-protein containing an F-box motif; (d) a peptide comprising the binding domain of Cdc34 that interacts with Cdc53 or the binding domain of Cdc53 that interacts with Cdc34; (e) a peptide comprising the binding domain of Cdc53 that interacts with Skp1 or the binding domain of Skp1 that interacts with Cdc53; or, (f) inhibitors or enhancers of the interaction of Cdc34 or Skp1, with Cdc53.
The invention still further provides a method for identifying a substance that binds to a complex comprising an E2 ubiquitin conjugating enzyme and a protein of the Cullin family, a complex comprising an E2 ubiquitin conjugating enzyme, a protein of the Cullin family, an F-box binding protein, and optionally a protein containing an F-box motif, or a complex comprising a protein of the Cullin family and an F-box binding protein, comprising: (a) reacting the complex with at least one substance which potentially can bind with the interacting molecules in the complex, under conditions which permit the formation of conjugates between the substance and complex and (b) assaying for conjugates, for free substance, or for non-conjugated complexes. The invention also contemplates methods for identifying substances that bind to other intracellular proteins that interact with the complexes of the invention.
Still further the invention provides a method for evaluating a compound for its ability to modulate ubiquitin dependent proteolysis. For example a substance which inhibits or enhances the interaction of the molecules in a complex of the invention, or a substance which binds to the molecules in a complex of the invention may be evaluated. In an embodiment, the method comprises providing a known concentration of a complex of the invention, with a substance which binds to the complex, and a test compound under conditions which permit the formation of conjugates between the substance and complex, and removing and/or detecting conjugates.
The present invention also contemplates a peptide of the formula I which interferes with the interaction of Cdc53 and Skp1.
X1-Tyr-Met-X2-X3-Tyr-X4-X5-X6-Ty-X7-X8-Cys-X9 (SEQ ID NO: 48)
wherein X1 represents one to ten amino acids, X2 represents Met, Arg, Thr, or Glu, X3 represents Leu, Phe, or Val, X4 represents Asp or Thr, X5 represents Ala, Ser, His, or Thr, X6 represents Ile or Val, X7 represents Asn or Asp, X8 represents Tyr, Ile, or Met, and X9 represents Thr, Val, or Ala.
In an embodiment of the present invention a peptide of the formula Ia which interferes with the interaction of Cdc53 and Skp1 is provided:
X1-X2-X3-X4-X5-X6-Tyr-Met-X7-X8-Tyr-X9X10-X11-Tyr-X12-X13-Cys-X14 (SEQ ID NO: 49)
wherein X1represents Ile, Asn, His, Ser, or Ala, X2 represents Leu, Met or Phe, X3 represents Ser, Ala, Thr, or Asp, X4 represents Pro, Lys, Arg, or Ser, X5 represents Thr, Lys, Ser, or Glu, X6 represents Met, Asp, Tyr, Gln, or Arg, X7 represents Met, Arg, Thr, or Glu, X8 represents Leu, Phe, or Val, and X9 represents Asp or Thr, X10 represents Ala, Ser, His, or Thr, X11 represents Ile or Val, X12 represents Asn or Asp, X13 represents Tyr, Ile, or Met, and X14 represents Thr, Val or Ala.
The invention also relates to truncations and analogs of the peptides of the invention. The invention also relates to the use of a peptide of the formula I or Ia to interfere with the interaction of a protein of the Cullin family preferably Cdc53 and an F-box binding protein preferably Skp1; and, pharmaceutical compositions for inhibiting the interaction of a protein of the Cullin family preferably Cdc53 and an F-box binding protein preferably Skp1.
Further, the invention relates to a method of modulating the interaction of Cdc53 and Skp1 comprising changing the amino acid Tyr at position 48 and/or Met at position 49 in Cdc53.
The peptides and antibodies of the invention, and substances and compounds identified using the methods of the invention may be used to modulate ubiquitin dependent proteolysis, and they may be used to modulate cellular processes of cells (such as proliferation, growth, and/or differentiation, in particular glucose and methionine biosynthesis, gene expression, cell division, and transcription) in which the compounds or substances are introduced.
Accordingly, the antibodies, peptides, substances and compounds may be formulated into compositions for adminstration to individuals suffering from a proliferative or differentiative condition. Therefore, the present invention also relates to a composition comprising one or more of a peptide or antibody of the invention, or a substance or compound identified using the methods of the invention, and a pharmaceutically acceptable carrier, excipient or diluent. A method for modulating proliferation, growth, and/or differentiation of cells is also provided comprising introducing into the cells a peptide or antibody of the invention, a compound or substance identified using the methods of the invention or a composition containing same. Methods for treating proliferative and/or differentiative disorders using the compositions of the invention are also provided.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.